Excess synthesis and storage of triacylglycerol (TAG) underlie much of the pathophysiology of major chronic disorders, including obesity, hepatic steatosis, atherosclerosis, the metabolic syndrome, and diabetes. Non-alcoholic fatty liver disease is a frequent prelude to fibrosis, cirrhosis and hepatocellular carcinoma, and the presence of TAG droplets in liver is required for replication of viruses like hepatitis C. Our long term goal is to determine how the enzymes in the pathway of TAG synthesis contribute to hepatic fatty acid use and how lipid intermediates of the TAG pathway control hepatic pathologies. The pathway of TAG and phospholipid biosynthesis is initiated by the rate-limiting enzyme, glycerol-3-phosphate acyltransferase (GPAT). As many as four independent GPAT isoforms are expressed in every cell, but why are multiple isoforms needed? We hypothesize that, although each GPAT isoform can, in theory, initiate the entire pathway of glycerolipid synthesis, independent isoforms are required to direct fatty acids towards complex lipid synthesis or away from -oxidation, to alter the rate of TAG synthesis under differing physiological conditions, to adjust the fatty acid composition of phospholipids and TAG, and to regulate nutrient-mediated signaling cascades that involve mTOR, PPAR?, and protein kinase C. To investigate how enhanced TAG synthesis in liver and muscle links to insulin resistance; we will determine mechanism(s) that underlie the effects of glycerolipid products of GPAT isoforms on PPAR? target genes and on the insulin signaling pathway. We will also determine how GPAT4 functions in phospholipid synthesis and energy metabolism by studying Gpat4-/- mice. These mice exhibit poor growth, are metabolically inefficient, have abnormal kinetics of dietary lipid absorption, and show increased energy expenditure despite decreased activity. These studies, using unique mouse models and innovative approaches, will address major gaps in our understanding of why excess tissue TAG is intimately associated with insulin resistance. PUBLIC HEALTH RELEVANCE: This project will enhance our understanding of non-alcoholic fatty liver disease, fatty heart disease, and the metabolic syndrome by a) examining how the enzymes that initiate triglyceride (fat) synthesis direct fatty acids towards different downstream pathways and b) investigating the metabolites that link obesity, triglyceride synthesis, and signaling pathways that alter the liver's response to insulin and other hormones.